EP0952773B1 - Verfahren und zusammensetzung zur vorbeugung und behandlung von mit clostridium difficile assoziierten erkrankungen - Google Patents

Verfahren und zusammensetzung zur vorbeugung und behandlung von mit clostridium difficile assoziierten erkrankungen Download PDF

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EP0952773B1
EP0952773B1 EP96935833A EP96935833A EP0952773B1 EP 0952773 B1 EP0952773 B1 EP 0952773B1 EP 96935833 A EP96935833 A EP 96935833A EP 96935833 A EP96935833 A EP 96935833A EP 0952773 B1 EP0952773 B1 EP 0952773B1
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toxigenic
difficile
strain
strains
group
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EP0952773A4 (de
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Dale N. Gerding
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/12—Antidiarrhoeals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/33—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Clostridium (G)

Definitions

  • This invention relates to methods and compositions useful to prevent and treat Clostridium difficile -associated diseases.
  • CDAD Clostridium difficile -associated diseases
  • C. difficile -related diseases cause great economic losses.
  • young foals are extremely susceptible, infection generally resulting in death; chinchillas are also vulnerable.
  • Antibiotic treatment in animals is not only expensive, but is not completely effective. There is a need for relatively inexpensive and effective prophylactic methods and treatments.
  • Saccharomyces boulardii a yeast, which is reported to show some benefit in reducing relapses and in preventing antibiotic-associated diarrhea, but is not effective against C. difficile diarrhea (McFarland et al., 1994; Surawicz et al., 1989).
  • Saccharomyces boulardii a yeast, which is reported to show some benefit in reducing relapses and in preventing antibiotic-associated diarrhea, but is not effective against C. difficile diarrhea (McFarland et al., 1994; Surawicz et al., 1989).
  • a problem with Saccharomyces is that twice daily administration is required for four weeks, making compliance difficult for patients.
  • the invention relates to the use of a non-toxigenic strain of C. difficile selected from the group consisting of M3, M23 and T7 as classified on the basis of its restriction endonuclease pattern on an agarose gel according to the method described in Clabots et al., J. Clin. Microbiol., vol. 31, 1993, pages 1870-1875 for the manufacture of a medicament for administration to a subject, wherein the non-toxigenic strain of C. difficile is administered after inhibition of antibiotic treatment in an amount sufficient to establish colonization of the gastrointestinal tract of a subject to prevent C. difficile associated disease.
  • said strain is a combination of two or more non-toxigenic strains.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a non-toxigenic strain of C. difficile selected from the group consisting of M3, M23 and T7 as classified on the basis of its restriction endonuclease pattern on an agarose gel according to the method described in Clabots et al., J. Clin. Microbiol., vol. 31, 1993, pages 1870-1875 said strain being in an amount sufficient to establish colonization of the gastrointestinal tract of a subject to prevent C. difficile associated disease, and a pharmaceutically acceptable carrier.
  • said non-toxigenic T strain is a combination of non-toxigenic strains selected from the group consisting of M3, M23 and T7 strains.
  • this composition is formulated in a unit dosage form.
  • This invention also relates to a method for selecting a non-toxigenic strain of C. difficile that is effective in preventing C. difficile related disease, said method comprising:
  • the strain selected is relatively frequent within its group.
  • the invention relates to the use of one or more strains of non-toxigenic C. difficile selected from the group consisting of M3, M23 and T7 as classified on the basis of its restriction endonuclease pattern on an agarose gel according to the method described in Clabots et al., J. Clin. Microbiol., vol. 31, 1993, pages 1870-1875 in an amount which is effective to suppress the population of toxigenic C. difficile in the gastrointestinal tract of a patient in the manufacture of a medicament to control diarrhea in the patient.
  • non-toxigenic T strain is a combination of non-toxigenic strains selected from the group consisting of M3, M23 and T7 strains.
  • compositions for preventing and treating CDAD C. difficile -associated disease
  • subjects including humans and non-human animals, e.g. mammals and birds are described.
  • Subjects are persons or animals who have received antimicrobials or antineoplastics (which also have antimicrobial activity).
  • a composition of a selected non-toxigenic strain of C. difficile is administered within about 24 hrs. after antibiotic (antimicrobial) or antineoplastic agents. This is to allow time for suppression of the normal intestinal flora, but not enough time for toxigenic strains of C. difficile to become established in the gastrointestinal tract.
  • types of antimicrobial agents known to cause risk of C. difficile disease in human and non-human animals or birds include cephalosporins, clindamycin, ampicillin, tetracyclines.
  • antibiotics usually vancomycin or metronidazole
  • the compositions are administered.
  • the methods comprise administering to the subject an effective amount of spores (generally in the order of 5 x 10 5 to 10 6 colony-forming units) of a selected non-toxigenic strain of C. difficile . Higher or repeated doses may be required for treatment than for prevention. "Effectiveness" is determined by clinical criteria showing the risk of developing CDAD is reduced by 80% or more compared to comparably exposed patients or animals in the same environment.
  • compositions for use in the method including selected non-toxigenic strains of C. difficile .
  • Certain non-toxigenic strains (types) of C. difficile are found to prevent disease better against specific toxigenic types of C. difficile than against other toxigenic types. Methods to select these strains are described.
  • a suitable non-toxigenic strain is a strain selected from the M, T, C, AP or other non-toxigenic groups as described by Clabots et al. (1993) or as genetically engineered to be non-toxigenic. These groups are selected because their relative high frequencies as isolates from persons or humans in which colonization has occurred, predicts their success as colonizers for purposes of the invention. Generally, "relatively frequent" groups occur in at least 5% of the non-toxigenic isolates, preferably in at least 15%; and more preferably in at least 25% of the isolates. Similarly, within a group, preferred strains are those that are most frequent.
  • a single purified strain of the M group, or of another single group, or a combination of strains within a group or a combination of strains from different groups, are suitable.
  • M group strains, in particular M3 and M23 are preferred singly or in combination with other strains.
  • pharmaceutical compositions and unit dosage forms comprising a strain selected from the non-toxigenic C. difficile groups, or a combination of strains.
  • This strategy is based on the assumption that the two or more strains each colonize effectively when administered together and at the same time (so that neither strain has the advantage of being the first to arrive in the GI tract).
  • Equivalent amounts of each type in the combination are a starting point for administration to prevent any one type from having an advantage in numbers. Ratios are adjusted if initial amounts are not effective.
  • non-toxigenic strains of C. difficile by removing or manipulating the two genes responsible for production of the C. difficile toxins, A and B, so as to create strains that no longer produce the toxins responsible for causing C. difficile -associated disease (CDAD).
  • CDAD C. difficile -associated disease
  • Such strains which prevent CDAD in the same manner as naturally non-toxigenic strains are disclosed herein.
  • Such strains would have to undergo extensive safety and efficacy trials in animals and humans to assure that they were safe, e.g. had not acquired any inadvertent virulence properties as a result of genetic engineering. They would also have to be shown to be efficacious as a preventive measure by the methods disclosed herein, and to not revert back to their original toxigenic state or reacquire the toxin genes.
  • the methods and compositions are useful to prevent and treat disease in humans or non-human animals, and are particularly useful for preventing and treating multiple relapses.
  • the invention relates to the use of a non-toxigenic strain of C. difficile selected from the group consisting of M3, M23 and T7 as classified on the basis of its restriction endonuclease pattern on an agarose gel according to the method described in Clabots et al., J. Clin. Microbiol., vol. 31, 1993, pages 1870-1875 for the manufacture of a medicament for administration to a subject, wherein the non-toxigenic strain of C. difficile is administered after inhibition of antibiotic treatment in an amount sufficient to establish colonization of the gastrointestinal tract of a subject to prevent C. difficile associated disease.
  • said strain is a combination of two or more non-toxigenic strains.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a non-toxigenic strain of C. difficile selected from the group consisting of M3, M23 and T7 as classified on the basis of its restriction endonuclease pattern on an agarose gel according to the method described in Clabots et al., J. Clin. Microbiol., vol. 31, 1993, pages 1870-1875 said strain being in an amount sufficient to establish colonization of the gastrointestinal tract of a subject to prevent C. difficile associated disease, and a pharmaceutically acceptable carrier.
  • said non-toxigenic T strain is a combination of non-toxigenic strains selected from the group consisting of M3, M23 and T7 strains.
  • this composition is formulated in a unit dosage form.
  • This invention also relates to a method for selecting a non-toxigenic strain of C. difficile that is effective in preventing C. difficile related disease, said method comprising:
  • the strain selected is relatively frequent within its group.
  • the invention relates to the use of one or more strains of non-toxigenic C. difficile selected from the group consisting of M3, M23 and T7 as classified on the basis of its restriction endonuclease pattern on an agarose gel according to the method described in Clabots et al., J. Clin. Microbiol., vol. 31, 1993, pages 1870-1875 in an amount which is effective to suppress the population of toxigenic C. difficile in the gastrointestinal tract of a patient in the manufacture of a medicament to control diarrhea in the patient.
  • non-toxigenic T strain is a combination of non-toxigenic strains selected from the group consisting of M3, M23 and T7 strains.
  • C. difficile -associated diseases CDAD
  • Prevention is used herein to mean that the risk of developing CDAD is reduced by approximately 80% or more compared to comparably exposed animals or humans in the same environment. Prevention is achieved by administering prophylactically a non-toxigenic strain or combination of strains of C. difficile to a subject at risk.
  • a subject at risk is one who has received antimicrobial or antineoplastic agents having antimicrobial activity, and is in a high-risk environment such as a hospital or nursing home for humans; or a flock or herd of animals where disease due to C. difficile is occurring or likely to occur due to administration of antibiotics.
  • CDAD The usual presentation of CDAD includes the presence of: (1) diarrhea, defined by a variety of criteria (e.g., at least six watery stools over 36 hours, three unformed stools in 24 hours or 2 days, or eight unformed stools over 48 hours); (2) pseudomembranes seen at lower gastrointestinal endoscopy, or toxin A or B from C. difficile detected in the stool, or a stool culture positive for the presence of a toxin-producing C. difficile ; and (3) no other recognized etiology for the diarrhea. (Gerding, 1995)
  • a method comprises administering to a subject a non-toxigenic strain of C. difficile preferably a strain selected from one or more of the M, T, C, P, S and AP groups.
  • the M group is a group of C. difficile strains identified by restriction endonuclease analysis (REA) of the total genomic DNA of approximately 4000 C. difficile isolates. Most of the isolates were obtained from patients and environmental sources at the Minneapolis Veterans Affairs Medical Center over a period of ten years. The remaining isolates were obtained from various other hospitals. The method in which the REA typing of these isolates was performed is described in Clabots et al. (1993).
  • the C. difficile isolates were classified into groups on the basis of DNA restriction band pattern similarity on agarose gels. Briefly, the first isolate with a new DNA band pattern was arbitrarily designated as a reference REA type. Similarities between new and reference REA types were scored by visual comparison of each 1-mm segment of the top 60 mm of the DNA band patterns run on the same gel. A similarity index (SI) was calculated as the number of identical segments expressed as a percentage of the total segments. Any new REA type with an SI of ⁇ 90% compared with an existing reference REA type was included in that group and given a specific type designation, for example, in the "M" group, types are designated as M1, M2, M3 .... M23 .... M35. Any new REA type with an SI of ⁇ 90% was designated the primary reference REA type for a new group and was used for future group comparisons. The groups were designated by letters, and distinct REA types within a group were designated by arabic numbers.
  • the group arbitrarily designated "M.” contains the largest number of isolates (347 of the 4000 toxigenic and non-toxigenic isolates typed so far), wherein "isolates” means an independently obtained specimen of the organism.
  • the members of the M group are closely related, with an SI of over 90% by definition.
  • the M group contains 35 distinct REA types (members of an REA type have an SI of 100%), with M3 and M23 being the most prevalent REA types. All isolates of the M group are non-toxigenic.
  • the size of the group is not limited to 35 because new M types are identified from time to time.
  • Strains M3 and M23 are particularly preferred strains. They have been found to prevent CDAD in 95-100% of hamsters challenged acutely with toxigenic strains of C. difficile known to be highly virulent in humans and 100% fatal in hamsters (see Example 1). The M strains are more protective in hamsters than previously tested strains, for which survival only improved by 72% (Wilson and Sheagren, 1983, Boriello and Barclay, 1985), and was not as durable as with M strains. M3 prevents CDAD for at least 60 days after its administration, and M3-treated animals remain disease free for at least 100 days after challenge with highly virulent toxigenic strains (see Example 1). The very high level of protection and the fact that the protection is extremely durable were not to be expected in view of the results reported in the prior art.
  • Groups are stored at the VA Lakeside Medical Center, 333 East Huron, Chicago, Illinois 60611 as a library of more than 75 distinct C. difficile REA groups including the M, B, J, C, AP, P, S, and T groups.
  • FIG. 1 illustrates the frequency of isolation of non-toxigenic C. difficile REA types for which more than 3 isolates were found.
  • C. difficile Strains of C. difficile are cultured and identified as C. difficile by standard microbiological methods well known in the art. For suitable techniques, see Clabots et al., (1993) and Kristjansson et al. (1994). C. difficile concentration is measured in colony forming units (cfu), as is well known in the art.
  • FIG. 2 shows representative agarose gels of Hind III-restricted DNA from Clostridium difficile strain type M1 (reference strain for the M group), and strains M3, M23, M4, M2, M14 (the most frequently isolated M types in descending order of frequency of isolation), strain VPI 2018 used by Wilson and Sheagren and not an M group strain, and strain type J9, a toxigenic C. difficile strain shown for comparison.
  • Molecular weight markers of lambda DNA restricted with Hind III are shown on the left and right lanes of the gel.
  • FIG. 3 is similar to FIG. 2 in showing gels of M3, M4, M23, J9 and VPI 2018, and in addition shows two other non-toxigenic types, T7 and S1.
  • M3 or other M type isolates By use of an M3 or other M type isolates, other M3 isolates can be identified by their identical restriction endonuclease band pattern or DNA fingerprint. Similar procedures are followed for the other groups.
  • Subjects at risk for CDAD include humans and animals receiving antibiotics or antineoplastic drugs, especially the elderly and those who are hospitalized for prolonged periods of time or institutionalized.
  • Strains of C. difficile from the M group are useful, because of their ability to colonize well. Such strains are also useful for therapeutic purposes to treat patients who have had relapses of C. difficile diarrhea following treatment with antibiotics such as vancomycin or metronidazole. Such patients are exceedingly difficult to cure and are preferred choices for administration of the non-toxigenic strains.
  • Such therapy is preferably administered after first treating the patient with vancomycin or metronidazole to reduce the population of toxigenic C. difficile and to control diarrhea. The vancomycin or metronidazole is stopped for 12-24hr, and then the non-toxigenic strain of C. difficile is administered orally in the same manner as described for the preventive use of the strain.
  • non-toxigenic C. difficile may be required for treatment as compared to prevention of CDAD.
  • Strains M, T, C, AP, P, S and combinations thereof are suitable.
  • M strains of C. difficile are preferred for treatment of relapses because of the known high rate of colonization by M strains in humans, and the low rate of C. difficile disease in humans colonized by non-toxigenic strains of C. difficile .
  • M strains for prevention or treatment of animals are also suitable, particularly in the horse and chinchilla industry where disease is common and frequently fatal.
  • Use of the M strains in animals for prevention or treatment of C. difficile disease is also within the scope of the invention.
  • non-toxigenic strain of C . difficile is administered to the subject prior to the patient's developing CDAD.
  • the non-toxigenic strain of C. difficile is administered shortly after the patient begins to receive antibiotics, especially if other patients in the same facility have developed CDAD.
  • the timing of administration to patients who have received antibiotics is critical. Colonization is unlikely to occur if antibiotics have not been given, because the normal bacterial flora of the gastrointestinal (GI) tract will prevent colonization.
  • GI tract flora permit colonization.
  • protective colonization should be accomplished within 24-72 hours of antibiotic initiation in order to achieve protection before exposure to toxigenic strains occurs.
  • the non-toxigenic strain may be administered at any time following initiation of antibiotic treatment.
  • the non-toxigenic strains of C. difficile are administered orally or enterally to a subject via, e.g. a nasogastric or gastric tube.
  • Effective dosage forms and dosage amounts effective to prevent CDAD may be determined empirically, and making such determinations is within the skill of the art. It is understood by those skilled in the art that the dosage amount will vary with the particular M or other group strain used, as disclosed herein, the patient's status (including illnesses being treated, age and size), other drugs including antibiotics being administered to the patient, and other factors well known in the medical art to affect dosage. Amounts and numbers of doses required for successful treatment may be higher than required for prevention.
  • a dosage of non-toxigenic C. difficile is selected that is sufficiently high to colonize the GI tract of the recipient, for example, 10 4 to 10 6 cfu per kg of body weight are suitable. Exact dosages required in humans are refined by studies in normal human volunteers. Multiple doses or larger doses may be required for certain strains and in certain situations such as when the patient is receiving ongoing antimicrobials (because the antimicrobial being given may inhibit or kill the C. difficile being given).
  • Exemplary dosages are those within the range of from about 10 4 to about 10 8 cfu per kg, preferably 10 4 to about 10 6 cfu per kg. Higher doses or repeated administration may be required if colonization is not established with the first dose administered. A single dose is expected to establish colonization, but the concomitant administration of certain antimicrobials to which the non-toxigenic C. difficile is susceptible may require administration of additional doses.
  • the non-toxigenic strain is administered as a pharmaceutical composition
  • a pharmaceutical composition comprising the non-toxigenic strain of C. difficile in combination with a pharmaceutically-acceptable carrier.
  • a pharmaceutically-acceptable carrier Each carrier must be "acceptable” in the sense of being compatible with the non-toxigenic strain, such as saline or glucose solution, should also not be injurious to the patient and should not kill or inhibit growth of the micro-organisms in the composition.
  • the amount of the strain that will be combined with a carrier to produce a unit dosage form will vary depending upon the factors described above.
  • the amount will generally be that amount which is the lowest dose effective to produce colonization and a preventive or therapeutic effect. Such determination is made by conducting trials in normal volunteers who first receive an antimicrobial such as clindamycin followed at 24-48h by the oral non-toxigenic C. difficile strain to be tested.
  • Formulations of the invention suitable for oral administration include those in the form of capsules, cachets, pills, tablets, powders, granules, or a suspension in an aqueous liquid, each containing a predetermined amount of a strain either as vegetative cells or spores. Spores are the preferred state of the organism for administration because they survive drying, lyophilization and exposure to air.
  • Methods to prepare spores of C. difficile include growth in a liquid or a solid medium to induce sporulation (Wilson et al., 1992). Quantitation of the number of spores present is determined by culture of 10-fold dilutions of the spore suspension under anaerobic incubation conditions.
  • the liquid dosage form may contain inert carriers, commonly used in the art, such as, for example, water, buffers or other aqueous solvents.
  • the liquid dosage form can also include adjuvants such as suspending, sweetening, flavoring, and coloring agents. [e.g. a glucose solution, fructose solution.] It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like in the liquid dosage form.
  • Another preferred dosage form is a powder or granules which can be reconstituted into a liquid dosage form just prior to use by the addition of water or a similar diluent.
  • the formulations are suitably presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials.
  • the formulations may be stored in a lyophilized condition and be administered with the addition of the sterile liquid carrier, for example water for injection, immediately prior to use.
  • the lyophilized formulations may also be administered in capsule form without the use of a liquid carrier.
  • Extemporaneous suspensions may be prepared from sterile powders, granules and tablets.
  • Human volunteers will be administered an antimicrobial followed by non-toxigenic C. difficile to determine if the gastrointestinal tracts of the volunteers become colonized, the dosage required to do so, the duration of colonization, and the safety/side effects of colonization. If these studies show that the volunteers are safely colonized for a sufficient period of time (probably one or more months), then a strain or combination of strains will be selected for a prevention trial in patients.
  • the prevention trial in patients will be conducted at multiple hospitals where patients who are beginning antibiotic treatment will be asked to participate in a double-blinded, placebo-controlled trial to determine if C. difficile disease can be prevented by colonizing the patients with non-toxigenic C. difficile . Hospitals will be selected that have high rates of CDAD in their institutions. End points will be 1) number of CDAD cases in treated versus placebo patients, 2) success in colonizing patients with non-toxigenic C. difficile , 3) adverse or side effects in treated vs placebo patients.
  • EXAMPLE 1 Using Strain M3 to Prevent CDAD In Hamsters
  • the Syrian hamster model is by far the most frequently used and best described model of CDAD in an animal. It is considered predictive of results in humans, although the disease is more severe and more often fatal in hamsters than in humans. (Onderdonk, 1988)
  • Clindamycin was administered to groups of ten of the hamsters by oral gavage needle at a dose of 30 mg/kg. Five days after receiving the antibiotic, the hamsters were given spore preparations of toxigenic C. difficile B1 or J9 by oral gavage. The spore preparations were prepared according to the method of Wilson 1982 and 1983, but were administered in a single aliquot of 100 cfu per animal. This dose had previously been determined to be lethal in dose-ranging studies.
  • the toxigenic strains used were REA types B1 and J9, identified by their distinct restriction endonuclease pattern. (Clabots et al., 1993). These two strains were chosen because they cause epidemics of CDAD in human patients in hospitals. These strains are highly virulent, producing CDAD in nearly 50% of the patients from whom they were isolated.
  • Non-toxigenic REA type M3 was administered by oral gavage to a group of 10 hamsters 48 hours after treatment with clindamycin as described above in section A.
  • the oral inoculum was a spore preparation (prepared as described in section A) containing a large number of spores (5 x 10 5 to 10 6 cfu per animal) in order to achieve colonization with a single dose.
  • Feces were cultured using TCCFA medium. All hamsters had high counts of M3 in fecal pellets by 28 to 72 hours post inoculation with the spores.
  • Feces were cultured using TCCFA medium.
  • the remaining five hamsters were challenged 60 days after receiving the protective strain M3 with 100 cfu per animal of toxigenic C. difficile strain B1 by oral gavage as described in Section B. Although M3 could no longer be detected in the stool pellets of these animals, they remained well and developed no ill-effects as a result of the B1 challenge. The hamsters were followed for an additional 92-100 days following B1 challenge and developed no evidence of illness. At postmortem examination, there was no evidence of cestis or colitis. M3 was isolated from the cecum of one animal 159 days after M3 inoculation, which was 117 days after the last positive fecal pellet culture for M3.
  • M3 the protection afforded by M3 is extremely durable, as the hamsters were completely strains B1 and J9 for 54-60 days after becoming colonized with strain M3.
  • toxin A and probably the production of toxin B (the two toxins are almost invariably present together) are the most important virulence factors in C. difficile . (Barriello et al., 1990; Lyerly et al., 1988). Strain M3 was tested to determine if it produced toxins A and B or contained the genes for these two toxins.
  • M3 was found to be noncytotoxic, indicating the absence of Toxin B.
  • Southern hybridization was used to determine if the genes coding for toxins A and B were present in M3.
  • a modification of the transfer techniques described by Southern (1975) was used to perform this analysis.
  • DNA from C. difficile M3 was extracted, purified, Hind III-digested, and separated by agarose gel electrophoresis as described in Clabots et al., (1993). Following electrophoresis, the DNA was hydrolyzed by acid depurination, denatured in alkaline buffer, then neutralized. The DNA was then transferred to a Zetabind synthetic membrane filter (AMF-Cuno, Meriden, Conn.) by capillary transfer in a high salt solution, the filter rinsed in low salt buffer, and dried at 80°C.
  • AMF-Cuno Meriden, Conn.
  • Toxin A and toxin B gene sequences were assayed using oligonucleotide DNA probes derived from the published sequences of each gene (Barroso et al., 1990; Dove et al., 1990).
  • a 19-mer oligonucleotide probe from the toxin A gene sequence that is repeated 5 times near the 3' end and a 20-mer sequence from position 503 near the 5' end of the toxin B gene were used to probe M3.
  • an extensive set of toxin A and toxin B probes obtained from restriction digests of cloned toxin A and B genes were used.
  • the probes were labelled with 32 P-deoxycytidine triphosphate by random priming, then hybridized to the DNA fixed to the filter using the methods of Sambrook et al. (1989). The hybridized bands were then visualized by autoradiography.
  • M3 does not produce toxins A and B and does not contain the genes coding for these two toxins.
  • the absence of these virulence factors provides further confirmation that the administration of M3 to human patients is safe.
  • Non-toxigenic REA type M23 was administered by oral gavage at a dose of 10 6 cfu of spores to 10 Syrian Golden Hamsters 48 hours after treatment with clindamycin as described in Example 1, section A. Two hamsters that received clindamycin were not given M23 and served as unprotected controls.
  • Feces were cultured using TCCFA medium. All 10 hamsters that received M23 spores had C. difficile strain M23 detected in feces 50 to 72 hours after gavage administration. The two control hamsters had no C. difficile detected in feces.
  • Hamsters were then challenged on day 5 following the clindamycin treatment (3 days after administration of M23) with 100 cfu per animal of toxigenic strain B1 of C. difficile by oral gavage as described in Section B of Example 1. Diarrhea and mortality were monitored for a minimum of 28 days following B1 for all surviving animals. Feces were cultured using TCCFA medium on days 1, 2, 3 and weekly following challenge with the toxigenic isolate B1. As in Example 1, selective medium containing erythromycin was used to differentiate the mix of toxigenic challenge organisms (resistant to erythromycin) from the non-toxigenic protective organisms (sensitive to erythromycin) over time.
  • M23 colonized animal was found to have B1 in fecal cultures and died 48 hours after challenge with strain B1.
  • four of five (80%) of animals colonized with M23 were protected from rechallenge with toxigenic strain B1 after readministration of clindamycin.
  • Two uncolonized control animals died from B1 infection.
  • Post mortem examination showed typical hemorrhagic proliferation in all three animals that died.
  • Surviving animals were sacrificed 10 days following B1 challenge and showed no evidence of stiitis or colitis at postmortem examination.
  • strain M23 As was strain M3 and all types of the M REA group, strain M23 was found to be non-cytotoxic in the standard HEp-2 cell cytotoxicity assay and did not react in the TOX-A TEST for Toxin A (TOX-A TEST, TechLab, Blacksburg, VA). Molecular probing of M23 for the toxin A and toxin B genes was also negative.
  • a genetically engineered strains of non-toxigenic C. difficile are prepared by A. selecting a toxigenic strain of C. difficile ; B. deleting the genes that encode toxin A and toxin B, said deleting achieved by means of recombinant genetic methods. (The toxin A and B genes are identified and cloned).

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Claims (9)

  1. Verwendung eines nicht-toxigenen C. difficile-Stamms ausgewählt aus der Gruppe bestehend aus M3, M23 und T7 entsprechend der Klassifizierung auf der Basis von dessen Restriktionsendonuklease-Muster auf einem Agarose-Gel gemäß dem in Clabots et al., J. Clin. Microbiol., Band 31, 1993, Seiten 1870-1875 beschriebenen Verfahren zur Herstellung eines Arzneimittels für die Verabreichung an ein Subjekt, wobei der nicht-toxigene C. difficile-Stamm nach der Einleitung einer Antibiotikabehandlung verabreicht wird, in einer Menge die ausreicht, damit eine Kolonialisierung im Magen-Darm-Trakt eines Subjekts einsetzt, um eine C. difficile-assoziierte Krankheit zu verhindern.
  2. Verwendung nach Anspruch 1, wobei der Stamm eine Kombination aus zwei oder mehreren nicht-toxigenen Stämmen ist.
  3. Pharmazeutische Zusammensetzung, umfassend einen nicht-toxigenen C. difficile-Stamm ausgewählt aus der Gruppe bestehend aus M3, M23 und T7 entsprechend der Klassifizierung auf der Basis von dessen Restriktionsendonuklease-Muster auf einem Agarose-Gel gemäß dem in Clabots et al., J. Clin. Microbiol., Band 31, 1993, Seiten 1870-1875 beschriebenen Verfahren, in einer Menge die ausreicht, damit eine Kolonialisierung im Magen-Darm-Trakt eines Subjekts einsetzt um eine C. difficile-assoziierte Krankheit zu verhindern, und einen pharmazeutisch annehmbaren Träger.
  4. Zusammensetzung nach Anspruch 3, wobei der nicht-toxigene T-Stamm eine Kombination aus nicht-toxigenen Stämmen ausgewählt aus der Gruppe bestehend aus M3-, M23- und T7-Stämmen ist.
  5. Zusammensetzung nach Anspruch 3 oder 4 formuliert als eine Einheitsdosisform.
  6. Verfahren zur Selektion eines nicht-toxigenen C. difficile-Stamms der zur Vorbeugung einer C. difficile-bezogenen Krankheit wirksam ist, wobei das Verfahren umfasst:
    a) Isolieren eines C. difficile-Stamms von einem Subjekt in einer Population;
    b) Bestimmen, dass der Stamm weder Toxin A noch B hat;
    c) Bestimmen der relativen Häufigkeit der Gruppe zu der der isolierte Stamm von b) gehört durch dessen Restriktionsendonuklease-Muster auf einem Agarose-Gel;
    d) Auswählen eines Stamms der zu einer relativ häufigen Gruppe gehört, die in dem Subjekt anzutreffen ist, wobei häufig definiert ist als mindestens 5% von nicht-toxigenen Isolaten, die in der Population von Isolaten anzutreffen sind.
  7. Verfahren nach Anspruch 6, wobei der Stamm aus einer relativ häufigen Gruppe innerhalb von dessen Gruppe ausgewählt ist.
  8. Verwendung eines oder mehrerer nicht-toxigenen C.diffcile-Stämmen ausgewählt aus der Gruppe bestehend aus M3, M23 und T7 entsprechend der Klassifizierung auf der Basis von dessen Restriktionsendonuklease-Muster auf einem Agarose-Gel gemäß dem in Clabots et al., J. Clin. Microbiol., Band 31, 1993, Seiten 1870-1875 beschriebenen Verfahren, in einer Menge die ausreicht, um die Population von toxigenen C.difficile in dem Magen-Darm-Trakt eines Patienten zu unterdrücken, zur Herstellung eines Arzneimittels um Diarrhoe in dem Patienten zu kontrollieren.
  9. Verwendung nach Anspruch 8, wobei der nicht-toxigene T-Stamm eine Kombination von nicht-toxigenen Stämmen ausgewählt aus der Gruppe bestehend aus M3-, M23-, und T7-Stämmen, ist.
EP96935833A 1995-09-15 1996-09-13 Verfahren und zusammensetzung zur vorbeugung und behandlung von mit clostridium difficile assoziierten erkrankungen Expired - Lifetime EP0952773B1 (de)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9433651B2 (en) 2013-06-05 2016-09-06 Rebiotix, Inc. Microbiota restoration therapy (MRT), compositions and methods of manufacture
US9463208B2 (en) 2010-02-01 2016-10-11 Rebiotix, Inc. Bacteriotherapy for clostridium difficile colitis
US9511100B2 (en) 2013-06-05 2016-12-06 Rebiotix, Inc. Microbiota restoration therapy (MRT), compositions and methods of manufacture
US9511099B2 (en) 2013-06-05 2016-12-06 Rebiotix, Inc. Microbiota restoration therapy (MRT), compositions and methods of manufacture
US9694039B2 (en) 2013-06-05 2017-07-04 Rebiotix, Inc. Microbiota restoration therapy (MRT), compositions and methods of manufacture
US9782445B2 (en) 2013-06-05 2017-10-10 Rebiotix, Inc. Microbiota restoration therapy (MRT), compositions and methods of manufacture
US10226431B2 (en) 2015-06-09 2019-03-12 Rebiotix, Inc. Microbiota restoration therapy (MRT) compositions and methods of manufacture
US10799539B2 (en) 2015-06-09 2020-10-13 Rebiotix, Inc. Microbiota restoration therapy (MRT) compositions and methods of manufacture
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Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6969520B2 (en) 1997-10-20 2005-11-29 Acambis Inc. Active immunization against clostridium difficile disease
CN101500581B (zh) * 2006-06-08 2013-10-30 科内尔研究基金会 编码艰难梭菌毒素a和毒素b受体结合域的密码子优化dna分子及其使用方法
US20130224164A1 (en) * 2010-09-10 2013-08-29 Viropharma Incorporated Environmental Clostridial Bacteriotherapy and Related Formulations and Methods of Manufacture and Use
DK3549949T5 (da) 2011-04-22 2024-09-02 Wyeth Llc Sammensætninger vedrørende et mutant Clostridium-difficile-toksin og fremgangsmåder dertil
ES2792106T3 (es) * 2012-05-18 2020-11-10 Genome Res Ltd Métodos y grupos
BR122016023101B1 (pt) 2012-10-21 2022-03-22 Pfizer Inc Polipeptídeo, composição imunogênica que o compreende, bem como célula recombinante derivada de clostridium difficile
US8906668B2 (en) 2012-11-23 2014-12-09 Seres Health, Inc. Synergistic bacterial compositions and methods of production and use thereof
KR102617655B1 (ko) 2012-11-23 2023-12-27 세레스 테라퓨틱스, 인코포레이티드 상승작용적 박테리아 조성물, 그리고 이의 제조 방법 및 용도
EP2951283A4 (de) 2013-02-04 2017-01-25 Seres Therapeutics, Inc. Zusammensetzungen und verfahren
KR20230110367A (ko) 2013-02-04 2023-07-21 세레스 테라퓨틱스, 인코포레이티드 조성물 및 방법
EP2967077A4 (de) 2013-03-15 2016-09-14 Seres Therapeutics Inc Netzwerkbasierte mikrobielle zusammensetzungen und verfahren
US10383901B2 (en) 2013-06-05 2019-08-20 Rebiotix, Inc. Microbiota restoration therapy (MRT), compositions and methods of manufacture
US10258655B2 (en) 2013-11-25 2019-04-16 Seres Therapeutics, Inc. Synergistic bacterial compositions and methods of production and use thereof
WO2015095241A2 (en) 2013-12-16 2015-06-25 Seres Health, Inc. Bacterial compositions and methods of use thereof for treatment of immune system disorders
KR102554351B1 (ko) 2016-06-14 2023-07-13 베단타 바이오사이언시즈, 인크. 클로스트리디움 디피실레 감염의 치료
US9999641B2 (en) 2016-06-14 2018-06-19 Vedanta Biosciences, Inc. Treatment of clostridium difficile infection
HUE052258T2 (hu) * 2017-06-14 2021-04-28 4D Pharma Res Ltd Megasphaera nemzetségbe tartozó baktériumtörzset tartalmazó készítmény, és alkalmazása
KR102680943B1 (ko) 2017-08-14 2024-07-03 세레스 테라퓨틱스, 인코포레이티드 담즙정체성 질환 치료를 위한 조성물 및 방법

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SAMBOL S.P. ET AL: "Colonization for the prevention of Clostridium difficile Disease in Hamsters.", THE JOURNAL OF INFECTIOUS DISEASES, vol. 186, 2002, pages 1781 - 1789 *

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